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338
FORMULATION DEVELOPMENT AND CHARECTERIZATION OF SUBLINGUAL
FAST DISSOLVING KETOCONAZOLE NIOSOMAL FILMS
*K. Sowmya and Dr. G. Praveen Kumar
Pharmceutics Sahasra Institute of Pharmaceutical Sciences, Hasanparthy Mandal, vangapahad Village, Warangal,
Telangana.
Article Received on 06/01/2021 Article Revised on 26/01/2021 Article Accepted on 16/02/2021
INTRODUCTION
Rapid liquifying movies have actually recently brought
in enhancing passion in the pharmaceutical market as a
result of the enhanced patient conformity, accurate
dosing, rapid onset of activity, positive preference, in
addition to their hassle-free handling and
administration.[1-3]
These films consist of thin oral strips
developed utilizing hydrophilic polymers that swiftly
degenerate and dissolve when placed in the oral cavity to
release the drug, which becomes available for
oromucosal absorption, without chewing and also intake
of water.[2,4,5]
Hence, it provides a convenient way for
patients who can not be dosed by mouth like pediatric as
well as geriatric patients,[6]
as well as additionally for
individuals that are not able to swallow huge amount of
water, such as those experiencing dysphagia, repeated
emesis, nausea, and also mental illness.[7]
Nevertheless,
medications that require high doses can not be
incorporated into movie strip due to its minimal surface
area.[8]
A loading of 62.5 mg of simethicone per slim
strip was successfully attained by Novartis Consumer
Wellness's Gas-X ® (East Hanover, NJ, USA).For
systemic medication shipment, rapid dissolving movies
can be used through sublingual route,[9]
as a result of the
high vascularity as well as permeability of this area,
which permits rapid absorption as well as fast action of
the incorporated medication.[10]
In addition, sublingual
management stays clear of first-pass hepatic
metabolism.[11,12]
Hence, this path can be used to boost
oral bioavailability of medicines that undergo
considerable first-pass effect.[13,14]
SJIF Impact Factor 6.044 Research Article ejbps, 2021, Volume 8, Issue 3, 338-352.
European Journal of Biomedical AND Pharmaceutical sciences
http://www.ejbps.com
ISSN 2349-8870
Volume: 8
Issue: 3
338-352
Year: 2021
*Corresponding Author: K. Sowmya
Pharmceutics Sahasra Institute of Pharmaceutical Sciences, Hasanparthy Mandal, vangapahad Village, Warangal, Telangana.
DOI: https://doi.org/10.17605/OSF.IO/XP9K6
ABSTRACT
Ketoconazole is a drug used to treat fungal infections in pet dogs. It can additionally be made use of to deal with
Cushings illness. Niosomes were prepared and evaluated for their abilities to enhance systemic delivery of
Ketoconazole, as compared to administration of oral tablets. FTIR Studies revealed that excipients were
compatible with the drug. Pre-formulation study for the drug surfactant compatibility by FTIR gave conformation
about their purity and showed no interaction between drug and selected surfactant various formulations were
developed by using non ionic surfactants (cholesterol, span 60) these are thin film hydration. Developed niosomes
were evaluated for size and shape, surface morphology, drug entrapment efficiency, in vitro drug release studies,
stability properties. The best entrapment efficiency and the best in-vitro drug release profile were achieved by
formulations. it was concluded that, Ketoconazole was successfully encapsulated into niosomes. Several in vitro
and in vivo characterizations of both the drug-loaded niosomes and films were performed. Ketoconazole was first
entrapped in different niosomal formulations, and drug-loaded niosomes with small size, low polydispersity, and
high EE were selected for incorporation into different fast dissolving films, which were then evaluated for
different physical characteristics. The optimal niosomal film showed sustained release of the drug compared to the
medicated film containing the free drug. The in vitro release kinetics of drug from the niosomal suspension and
niosomal film followed the Higuchi diffusion model. Moreover, the in vivo study in rabbits showed significantly
higher rate and extent of Ketoconazole absorption from sublingual fast dissolving niosomal film compared to that
from oral commercial tablets. Consequently, the absolute bioavailability of the drug following sublingual
administration was significantly higher than that after oral tablet administration. These results indicated that the
prepared sublingual fast dissolving niosomal film could have potential as an efficient delivery system to enhance
the bioavailability and prolong the therapeutic effect of Ketoconazole, thus improving the patient compliance by
eliminating the need for frequent dosing of the drug.
KEYWORDS: Ketoconazole, Niosomal film, cholesterol, span 60.
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339
Ketoconazole is a drug used to treat fungal infections in
pet dogs. It can additionally be made use of to deal with
Cushings illness. The drug functions by obstructing
cortisol development in the adrenal glands and inhibiting
manufacturing of the fungal cell wall surfaces.
Ketoconazole is a common type of Nizoral. There are
other kinds of this drug that have been produced to lower
the number of adverse effects connected with
Ketoconazole. Itraconazole as well as Fluconazole are
two of these medications. Mechanism of Activity
Ketoconazole blocks the synthesis of ergosterol, an
essential element of the fungal cell membrane layer, with
the inhibition of cytochrome P-450 reliant enzyme
lanosterol 14α-demethylase responsible for the
conversion of lanosterol to ergosterol in the fungal cell
membrane.[15]
Figure 1: Chemical structure of Ketoconazole.
MATERIALS
Ketoconazole was a gift sample from Micro labs,
Hyderabad, India, Hydroxypropyl methyl cellulose are
from Colorcon Asia Pvt. Ltd., Goa, Polyethylene Glycol
400 from Lobachem, Mumbai, Standard chemical
reagents from SD fine chemical Ltd, Hyderabad.
Methanol was of high performance liquid
chromatography (HPLC) grade. All other reagents and
solvents were of analytical reagent grade
Methodology
Construction of standard curve
Scanning of ketoconazole
Ketoconazole 10mg of pure drug as dissolved in
methanol and diluted to give concentration 10mg and
was scanned between 200 – 400nm wavelength for the
determination of the wavelength of 269 nmwas selected
as for max.
The same used for future analysis of drug solution and
absorbance of final standard solution was measured at
269 nm.
Preparation of 6.8 pH phosphate buffer Dissolve 35.084 grs of di-sodium hydrogen phosphate
and 13.872 grs of potassium di-hydrogen orthophosphate
in sufficient water .to produce 1000 ml.
Construction of Standard Calibration Curve of
Ketoconazole
Principle: Ketoconazole exhibits peak absorbance at 269
nm in methanol.
Instrument used
shimadzu – 1800 ultra violet spectrophotometer, japan.
Procedure
Preparation of standard solution
Standard stock solution of Ketoconazole prepared by
dissolving 10mg of Ketoconazole in methanol to produce
a concentration of 1000μg/ml. 1ml of this stock solution
was taken then diluted up to 10ml by using methanol to
produce a concentration of 100μg/ml which is the
standard stock solution. From the above stock solution,
5ml was pipette out into a 10ml volumetric flask and the
volume was made up to the mark with methanol to
prepare a concentration of 50μg/ml. Then the sample was
scanned in UV-VIS Spectrophotometer in the range 400-
500nm using methanol as a blank and the wave length
corresponding to maximum absorbance (λmax) was
found to be 269nm.
Preparation of Working Standard Solution
0.5ml, 1ml, 2ml, 3ml, 4ml, 5ml of 100ug/ml solution was
diluted to 10ml using methanol to produce 5µg/ml,
10μg/ml, 50μg/ml, 30μg/ml, 40μg/ml, 50μg/ml solutions
respectively. The absorbance of each concentration was
measured at 269
Maximum = 269 nm
Compatibility Studies
IR spectroscopy can be used to investigate and
predictancy of physico-chemical interactions between
different components in a formulation and therefore it
can be applied to the selection of suitable chemically
compatible excipients.
The aim of present study was to test whether there is any
interaction between the carrier & drug. The IR
spectroscopy were recorder for following compounds.
Ketoconazole
Span 60
Cholesterol
Chloroform
Methanol
A small amount of sample was taken into mortar and
pestle triturated. The pellet was kept on to the sample
holder and scanned from 4000cm-1 to 400cm-1 in
Bruker IR – Spectrophotometer. Then it was compared
with original spectra. IR Spectra was compared and
checked for any shifting in functional peaks and
noninvolvement of functional group.
Design of experiments
32 A factorial design was adopted based on the
preliminary studies for the preparation of Ketoconazole
loaded niosomes. The concentration of span 60 (X1) and
cholesterol (X2) were chosen to study their effect on
percentage entrapment efficiency (Y1), In vitro drug
release at 8th
hour (Y2), vesicle size (Y3). The factors are
studied at three levels-1, 0 and 1 indicating low, medium
and high respectively. The statistical optimization
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340
procedure was accomplished with the help of Design-
Expert version 10. The software performs response
surface methodology including multiple regression
analysis, ANOVA and statistical optimization.
Preparation of ketoconazole loaded niosomes
Ketoconazole loaded niosomes were prepared using thin
film hydration method. Span 60 and cholesterol in
different ratios were dissolved in 10 ml of chloroform
and methanol mixture (2:1 v/v) in a round bottom flask.
100 mg of Ketoconazole was separately dissolved in 5
ml of chloroform and methanol mixture (2:1 v/v) and
added to surfactant mixture. The solvents were
evaporated under vacuum at 40°C in a rotary evaporator
(Buchi, Switzerland) at 120 rpm until a smooth and thin
film formed on the wall of the flask. After ensuring
complete removal of volatile solvents, hydration of the
surfactant film was carried out using 10 ml of distilled
water at 60°C ± 2°C with mechanical agitation to form a
niosomal suspension. The resulting niosomal suspension
was sonicated (Equitron, Mumbai) in 3 cycles of 1/1 min
on/off cycles leading to the formation of multilamellar
niosomes. The obtained niosomal suspension was left to
mature overnight at 2°C - 8°C and stored under
refrigeration for further studies.
Methods for Characterization of Niosomes
Determination of Entrapment Efficiency Niosomes containing Ketoconazole were separated from
free drug by cooling centrifugation at 15,000 rpm for 60
minutes at 4°C. The niosomal pellets were suspended in
methanol and centrifuged again.The integrity of vesicles
was not affected by centrifugation as reported in
literature. The washing procedure was repeated two
times as reported previously. The supernatant was
separated each time and assayed spectrophotometrically
at 269 nm. The amount of entrapped drug was obtained
by subtracting the amount of free drug from the total
drug. The percent of entrapment efficiency (EE%) was
then calculated according to Equation (each result is the
mean of three separate experiments).
EE% = Amount of entrapped drug / Total drug amount
×100
The particle sizes and zeta potential determination
The mean particle size (nm) and polydispersity index of
the prepared niosomes in both niosomal dispersion and
niosomal film were measured by dynamic light scattering
laser using a Zetasizer Nano ZS (Malvern Instruments,
Worcestershire, UK) equipped with a 4 mW helium/neon
laser (λ=633 nm) and thermoelectric temperature
controller. The corresponding zeta potentials (mV) were
determined by photon correlation spectroscopy using the
same Zetasizer Nano instrument
Preparation of Fast Dissolving Niosomal Films
Fast dissolving films were prepared by solvent casting
technique. HPMC and MC were used as film-forming
polymers. Polyethylene Glycol 400 was used as a
plasticizer, saccharine as a sweetener, and menthol as
flavoring agent and to give mouth refreshment feeling.
Concentrations of plasticizer, sweetener, and flavoring
agents were kept constant. Microcrystalline cellulose
(Avicel) was used as super disintegrant at two different
concentrations. Specified weight of film-forming
polymer was first dissolved in 20 mL of the casting
solvent (warm distilled water), and sweetener and
flavoring agent were dissolved in the polymeric solution.
The calculated amount of super disintegrant was
incorporated into the polymeric solutions after levigation
with the required volume of the plasticizer. For the
preparation of medicated films (containing free drug),
the required amount of Ketoconazole was directly added
and completely dissolved into the polymeric solution
before the addition of super disintegrant. For niosomal
film, a specified volume of the selected niosomal
dispersion (corresponding to the required Ketoconazole
dose) was incorporated and gently mixed with the
selected polymeric solution. The final volume was
adjusted to 25 mL with distilled water, and the beaker
was covered with aluminum foil to prevent solvent
evaporation. The casting solution was subjected to gentle
stirring for 2 hours using magnetic stirrer (L32; Bibby,
Staffordshire, UK). The casting solution (25 mL) was
transferred into a previously cleaned and dried Teflon-
coated plate (area =28 cm2, each 4 cm2 contains 25 mg
of drug). The solvent was allowed to evaporate for 72
hours, and the film was then removed from the Teflon
plate and was allowed to dry in a desiccator at least 48
hours before evaluation. The patches were punched into
4 cm2 pieces containing 25 mg of Ketoconazole, then
wrapped in an aluminum foil (to maintain the integrity
and elasticity of the films) and were finally stored in a
dry place at ambient room temperature. The films were
subjected to evaluation within 1 week of their
preparation.29
Composition of various formulations is
provided in Table 1.
Table 1: Composition of different Ketoconazole fast
dissolving films.
Component (mg) F1 F2 F3 F4 F5 F6
HPMc 90 90 90 0 0 0
Mc 0 0 0 100 100 100
Peg400 100 100 100 100 100 100
Saccharine 40 40 40 40 40 40
Menthol 16 16 16 16 16 16
Mcc 0 6 12 0 6 12
Evaluation of fast dissolving films
Physical and mechanical properties
The fast-dissolving films were evaluated for physical appear-
ance, surface texture, thickness, weight uniformity,
folding endurance, surface pH, and drug content
uniformity.
Physical appearance: The physical appearance was
checked with visual inspection of films and texture by
touch.
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Thickness: Thickness was measured by micrometer
screw gauge (Mitutoyo, Tokyo, Japan) at different points of
each formulation, and the mean values were calculated.
Weight variation: Weight variation was studied using
electronic Analytical balance (AJ150; Mettler, Greifensee,
Switzerland) by taking individual weights of ten
randomly selected 4 cm2 patches for each formulation
(prepared in different batches).
Disintegration time: In vitro disintegration time was
determined visually in a Petri dish containing 25 mL of
phosphate buffer (pH 6.8) with swirling every 10
seconds. The disintegration time is the time recorded
when the film starts to break or disintegrate. Folding
endurance was determined by repeated folding of the
film at the same place till the strip breaks. The number
of times the film is folded without breaking was
computed as the folding endurance value.
Determination of surface pH: For the determination of
surface pH, three films of each formulation were allowed
to contact with 1 mL of distilled water for 1 hour at room
temperature and measured by pH meter (Jenway,
Staffordshire, UK). The surface pH was measured by
bringing the electrode in contact with the surface of the
film and allowing it to equilibrate for 1 minute. Finally,
to check the uniformity of the drug content in the cast
film, 4 cm2 patches were cut from different places in cast
film, and each film was dissolved in 100 mL phosphate
buffer (pH 6.8). The resulting solution was filtered, and
further dilution was made with phosphate buffer and the
absorbance was mea- sured spectrophotometrically at
269 nm, and the percentage of the drug content was
determined. The same procedure was repeated for at least
three patches of each formulation, and the mean values
and standard deviations were calculated.
Fourier Transform Infrared Spectroscopy
The Fourier transform infrared spectroscopy (FTIR)
spectra of the selected niosomal film compared to its
corresponding physical mixture and the individual solid
components were recorded using FTIR
spectrophotometer (IR-470; Shimadzu, Kyoto, Japan).
Samples were mixed with potassium bromide
(spectroscopic grade) and compressed into disks using
hydraulic press before scanning from 4,000 to 400 cm-1
.
Differential Scanning Calorimetry
Differential scanning calorimetry (DSC) scans were
recorded for the selected niosomal film, its
corresponding physical mixture and the individual solid
components. The samples (3–5 mg) were hermetically
sealed in aluminum pans and heated at a constant rate of
100C /min, over a temperature range of 25
0C–200
0C.
Thermograms of the samples were obtained using DSC
(DSC-60; Shimadzu). Thermal analysis data were
recorded using a TA 50I PC system with Shimadzu
software programs. Indium standard was used to
calibrate the DSC temperature and enthalpy scale. N2
was used as purging gas at rate of 40 mL/min.
In-vitro drug release studies
The previously prepared film was removed from the
plate, cut in area of 4 cm2, and weighed on an analytical
balance. The release of Ketoconazole from either the
medicated niosomal dispersion or from the prepared
films (medicated films or niosomal films) was performed
in beakers each containing 100 mL of phosphate buffer
at pH 6.8 (beaker method). The beakers were placed in a
shaking water bath (Gesell- schaft für Labortechnik mbH,
Burgwedel, Germany) set at 370C and 100 rpm. Samples
were withdrawn (5 mL) at the determined time intervals
and were centrifuged, and then the supernatant was
filtered and assayed using a UV-visible
spectrophotometer (Shimadzu Seisakusho, Ltd., Kyoto,
Japan) at 269 nm. Samples were replaced by equal
volumes of fresh buffer to maintain the same volume in
the flasks. The experiment was conducted in triplicates.
The amount of drug released at each time interval was
calculated, and the cumulative amount of drug released
was calculated as a function of time to construct the drug
release profile graphs. The release data were kinetically
analyzed by curve fitting method to different kinetic
models of zero-order, first-order, Higuchi, and
Korsemeyer–Peppas models.
The result of in-vitro release profile obtained for all the
formulations for plotted in the modes of data treatment as
follows
Zero – order kinetic model cumulative % drug released
versus time
First - order kinetic model cumulative % drug release
versus time
Higuchi‟s model – cumulative percent drug released
versus square root of time
Zero Order Kinetics
Zero order release would be predicted by following
equation
At = AO - KOT
Where,
At = drug release at time t
Ao = initial drug concentration
Ko = zero order constant (hr – 1)
When the data is plotted as cumulative percent drug
release versus, time if the plot is linear then the data
obeys zero order kinetics and its slope equal to zero
order release constant Ko.
First Order Kinetics
First order release could be predicted by the following
equation:
Log C = Log Co – kot /2.303
Where,
C = amount of drug retained at t
Co = initial amount of drug
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K = First order rate constant (hr-1)
When the data plotted as log cumulative percent drug
remaining versus time, yields a straight line, indicating
that the release follow first order kinetics. The constant
K1 can be obtained by
Higuchi’s model
Drug release from the matrix devices by diffusion has
been described by following Higuchi‟s c; assical
diffusion equation:
Q = (DE/t(2A-ES) CST)1/2
Where,
Q = Amount of drug release at time „t‟
D = Diffusion coefficient of the drug in the matrix
A = Total amount of drug in unit volume of matrix
Cs = solubility of drug in matrix
E = Porosity of matrix
t = tortuosity $ time
Above equation can be simplified as if we assumes that
D, Cs and A are constant then equation becomes:
Q = K1/2
When the data is spited according to equation i.e
cumulative drug release versus square root of time yields
a straight line indicating.
That the drug released by diffusion mechanism. The
slope is equal to „k‟
Korsmeyar equation /pepa’s model
To study the mechanism of drug release from the
niosomal solution, the release data was also fitted to the
well-known exponential equation (Korsmeyar equation /
peppa;s law equation) which is often used to describe the
drug release behavior from polymeric systems.
Korsmeyer equation
Mt/Ma = Ktm
Where,
Mt/Ma = The fraction of drug released at time „t‟
K= Constant incorporating the structural and geometrical
characteristics of the Drug polymer system
N = Diffusion exponent related to the mechanism of the
release
Stability Study
For the prepared fast dissolving films, stability study was
car- ried out at two different storage conditions, one was
normal room conditions and the other was 400C /75%
relative humidity for 8 weeks. Each piece of the
conventional medicated film formulation (F6) and the
niosomal film (N4F6) were packed in butter paper
followed by aluminum foil and plastic tape. After 8 weeks,
the films were evaluated for the physical appearance,
surface pH, and in vitro drug release. Regarding the
prepared niosomal dispersion, stability study was carried
out at 40C, room temperature, and elevated temperature for
a period of 8 weeks. The samples of selected niosomal
disperion formulation (N4) were sealed in a glass vial and
stored at the selected temperature for 4 weeks. Samples
from each batch were withdrawn at definite time intervals
and evaluated for physical appearance, vesicle size, and
zeta potential, as compared to the reconstituted niosomal
dispersion after the niosomal film dissolution in
phosphate buffer (pH 6.8).
Scanning Electron Microscope
The surface morphology of the selected fast dissolving
niosomal film (N4F6) was observed and compared to
that of the selected niosomal dispersion formulation (N4)
using scanning electron microscope (JSM-5400 LV; Joel,
Japan) operated at an acceleration voltage of 15 kV. in
situ disintegration time and palatability studies.
A taste panel consisting of 14 healthy male volunteers,
nine males, and five females (25–45 years old) has tried
the selected niosomal film formulation (N4F6). Prior to
the study, the volunteers were briefed on the nature,
purpose, duration, and risk of the study. After explaining
the study, written informed consent was obtained from
all volunteers included. The tested film was kept in
mouth until disintegration. The volunteers were
requested to record the disintegration time, ease of
handling, and acceptance of the film and gave a score
based on the parameters, namely taste, after-taste,
mouth-feel refreshment, as presented in previously
reported studies.
Animal Dosing and Sampling Scheme
The plasma concentrations of Ketoconazole were
evaluated from healthy rabbits after the sublingual
administration of the selected niosomal films (N4F6)
compared to the commercial oral tablets, (100 mg
Ketoconazole). Fifteen healthy rabbits (1.5–2.25 kg)
were used in the present study. The rabbits were fasted
for 24 hours before the administration of the drug and
were anesthetized with 0.1 mL thiopentane (0.5 mg/mL).
Approximately 7 mg/kg of the drug, corresponding to a
100-mg human dose, was used. This equivalent dose for
rabbits was calculated by the aid of surface area ratio, as
the therapeutic dose of man was multiplied by a certain
mathematical factor obtained from a special table for
surface area ratios of some common laboratory species
and man. The following equation was used, Dr= Dh
(Wr/Wh),3/4 where Dr is the rabbit dose, Dh is the
human dose, Wr is the rabbit weight, and Wh is the
human weight. Rabbits were randomly divided into three
groups each of five animals as follows: the first group
received Ketoconazole commercial oral tablets by gastric
intubation, the second group was given the selected
Ketoconazole sublingual nio- somal film (N4F6), and the
third group was given intravenous Ketoconazole in
isotonic saline solutions. Control blood samples were
taken from the rabbits immediately before administration
of the drug. Multiple blood samples (1–2 mL) were
collected in heparinized vacutainer tubes before
administration and at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24
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343
hours following drug administration. The plasma was
then separated after centrifu- gation and stored frozen at
=200C until analysis.
Analysis of Plasma Samples
Ketoconazole plasma concentration was measured using
a reported using a sensitive high-performance liquid
chromatography assay. The high-performance liquid
chromatography system (Knauer K-500 pump, Knauer,
K-2500 UV detector, C-R6A, chromatopac integrator;
Shimadzu) was used with the reversed-phase mode.
Analysis was performed on Aqua RP-C18 packed
column (250±4.6 mm internal diameter, 5 mm particle
diameters). The 50-mL aliquots were injected and eluted
with a mobile phase containing 3.8 g ammonium acetate
in 810 mL water, 2 mL triethylamine, 10 mL glacial
acetic acid, 3 mL phosphoric acid, and 146 mL
acetonitrile.38
The flow rate was set at 1 mL/min, and the
eluent was monitored at 269 nm. A calibration curve of
Ketoconazole in the plasma was constructed using blank
plasma spiked with standard Ketoconazole solutions to
obtain a concentration range of 0.3–30 mg/mL. The
spiked plasma was then subjected to the same extraction
procedure of the samples. Triplicate runs were made for
each standard sample.
Pharmacokinetics Analysis
After measuring Ketoconazole concentrations in the
plasma, Ketoconazole pharmacokinetics was assessed by
fitting the plasma concentration–time data to the suitable
model using Win Nonlin standard version 1.5 software.
Absorption rate constant (Ka), absorption half-life (t½a),
elimination rate constant (Kel), elimination half-life (t½),
and the area under the plasma- Ketoconazole
concentration versus time curve (AUC) were calculated.
Also, the maximum concentration (Cmax) and the time
to reach the maximum concentration (Tmax) were
reported. The absolute bioavail- ability of the drug was
calculated by comparing the respective AUC after
extravascular and intravenous administration.
Statistical Analysis
Experiments were performed in triplicates unless other-
wise noted. Statistical assessment of differences between
experimental groups was performed by one-way analysis
of variance or two-sided Student‟s t-test for pairwise
comparison (GraphPad Prism 6.0; GraphPad, San Diego,
CA, USA). Differences between means were considered
statistically nonsignificant (NS) if the P-value was .0.05.
The parameters were taken as significantly (S) different
for 0.05.P$0.01 and highly significantly (HS)
different for 0.01.P$0.001.
8. RESULTS AND DISCUSSION
Analytical study
Solubility Studies of Ketoconazole
Water Very slightly soluble
Methanol Soluble
DMSO Soluble
PH 7.4 Buffer Sparingly Soluble
The availability of literature on solubility profile of
Ketoconazole is soluble in methanol, chloroform and pH
6.8 buffer and also it tells that the drugs solubility
increases by increasing the ph nearly alkaline. This was
confirmed by observing the solubility studies of
Ketoconazole practically. However pH 6.8 may be
suitable for diffusion studies ass sufficient solubility was
attained at this ph for Ketoconazole.
Scanning of Drug
Ketoconazole drug was scanned in methanol between
200 – 400nm using ultraviolet spectrophotometer was
identified its light absorbance pattern which follows the
absorption of light in the ranger 200 – 400nm and a
maximum absorbance was observed at 269nm. A peak
was obtained at about 269 nm which confirms the
Ketoconazole in presence. Ketoconazole spectrum gave a
highest peak at 269 nm and same was selected for further
evaluations.
Calibration curve In pH 6.8 buffer Standard solutions of different concentration were
prepared and their observation were measured at 269 nm
calibration curve was plotted against drug concentration
versus absorbance as given in figure below.
Table 2: Standard calibration curve.
Concentration Absorbance
0 0
2 0.183
4 0.367
6 0.523
8 0.684
10 0.876
Table 3: Parameters of Calibration Curve.
PARAMETERS Ketoconazole
WAVE LENGTH 269 nm
BEER’S LAW LIMIT 2 – 10
R2 VALUE 0.999
REGRESSION EQUATION 7.4 BUFFER Y = 0.086x + 0.008
R2 VALUE 0.999
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344
Figure 2: Scanning of Drug in 6.8 Ph Buffer.
Scanning of Drug in Methanol
Ketoconazole pure drug was scanned in methanol
between 200 – 400nm using ultraviolet
spectrophotometer Ketoconazole was identified its light
absorption pattern which follows the absorption of light
ranges 200 – 400nm and a maximum absorbance was
observed at 269 nm. A peak was obtained at about 269
nm was observed which confirms the Ketoconazole
presences.
Ketoconazole spectrum gave a highest peak 269 nm and
same was selected for future evaluations.
Calibration Curve in Methanol
Standard solution of different concentration were
prepared and their absorbance were measured at 269 nm
calibration curve was plotted against drug concentrations
versus absorbance as given in figure below
Table 4: Concentration and absorbance of Ketoconazole in methanol.
Concentration Absorbance
0 0
2 0.201
4 0.402
6 0.593
8 0.733
10 0.899
Figure 3: Absorbance of Methanol.
Ftir Studies (Fourier Transform Infra-Red
(Spectroscopy)
Fourier transform infrared spectroscopy (FTIR) is a
widely used analytical technique that is routinely applied
to the characterization of biomaterials. However,
preparing samples of biomaterials for infrared
spectroscopy is often a tedious process.
IR spectra was comparing and checked for any shifting
peaks and non- involvement of functional group. From
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the spectra inhibits clear that there is no interaction
between the selected carrier‟s drug and mixtures.
The main sampling problem in FTIR characterization of
biomaterials is that nearly all solid materials are too
opaque in their normal forms for direct transmission
analysis in the mid-infrared region.
Unfortunately, a limited amount of information is
available within the near-infrared spectral region,
whereas, the mid-infrared region provides most spectral
bands for the required characterization.
Ftir Studies of Cholesterol Cholesterol is an organic molecule. It is a sterol, a type
of lipid. Cholesterol is biosynthesized by all animal cells
and is an essential structural component of animal cell
membranes.
Figure 4: FTIR of Cholesterol,
Cholesterol wave number 1000 – 3500cm
Transmittance (%) 10 - 100
FTIR Studies of Span 60
Sorbitan tri-stearate is a nonionic surfactant. It is
variously used as a dispersing agent, emulsifier, and
stabilizer, in food and in aerosol sprays.
Figure 5: FTIR of SPAN 60.
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Wave number 1000 – 3500cm-1
Transmittance (%) 10 - 100
FTIR Studies of Ketoconazole Drug
Figure 6: FTIR of Ketoconazole drug.
Wave number 1000 – 3500cm-1
Transmittance (%) 10 - 100
Formulation of Ketoconazole
Figure 7: FTIR of Ketoconazole formulation.
Wave number 1000 – 3500cm-1
Transmittance 10 - 100
The FTIR spectra of the selected Ketoconazole nio-
somal film (N4F6), its corresponding physical mixture
and their individual solid components. FTIR studies were
performed to understand the compatibilities between the
drug with different excipients. The figures above
illustrate that the functional groups like N-H with the
observation range of 3350-3310 has peaks at 3283.32 in
pure drug and 3284.25 in optimized formulation. The
functional group C═O has a peak range of 1710-1680
has peaks at 1685.78 in pure drug and 1686.17 in
optimized formulation. Similarly the functional group C-
O has a peak range of 1310-1250 has peaks at 1289.80 in
pure drug and 1290.29 in optimized formulation. The
functional groups in both the pure drug and optimized
formulation are found. Hence it can be concluded that
the pure drug is compatible with the excipients used in
the study. IR ranges presented in table 5
Table 5: IR range of pure drug and optimized formulation.
Charecteristic peak Observed range Pure drug Optimized formulation
N-H 3350-3310 3283.32 3284.25
C═O 1710-1680 1685.78 1686.17
C-O 1310-1250 1289.80 1290.29
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Rheological Characterization
Scanning Electron Microscopy: SEM
The niosomal shape, size, and surface characteristics of
niosomes were observed by images. The SEM image of
Ketoconazole loaded niosomes after appropriate dilution
of with disteilled water respectively. The diameter of
niosomes were mainly nano in range to indicate the
discrete vesicular nature of niosomes with in gel matrix,
slight aggregates were observed in the SEM image
however no aggregates were detected on SEM image that
could be attributed to the preparation for capturing the
SEM images.
Figure of Scanning Electron Microscopy.
Figure 8: Scanning electron microscopy.
DSC Analysis: (differential scanning calorimeter)
Differential scanning calorimeter (DSC) experiments
were used to find out the presence of any interaction
among drug and the excipients and also find out the
whether there is any alteration of the drug. it quantities
the enthalpic changes during endothermic or exothermic
effects. the instrumented was calibrated with indium
(calibration standard, purity >99.99%) for melting point
and heat of fusion. Protein folding/unfolding reaction, as
any other chemical reaction, is accompanied by heat
effects. The heat of unfolding measured at a constant
pressure represents the enthalpy of the process. Direct
measurements of the heat of unfolding are done using
differential scanning calorimeter (DSC).
Picture of DSC (differential scanning calorimeter):
Sample Name: Ketoconazole Gas1: Nitrogen
Sample Weight: 1.15 mg Gas2: Oxygen
Reference Name: Aluminium
Reference Weight: 1.000 mg
Figure 9: DSC of Ketoconazole.
Transmission Electron Microscopy The TEM images confirmed the formation of niosomes.
The shapes of the vesicles were spherical, and they were
similar with the typical niosome micrographs obtained in
prior studies. The size of the niosomes was around the
average particle size measured by Zetasizer. The particle
size distribution histogram revealed the bimodal size
distribution of formulation. This was also confirmed by
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the TEM analysis demonstrating several niosomes
around 100 nm. But the percentage of larger niosomes
was observed to be very low compared to the general
size distribution.
Figure 10: Transmission electron microscopy (TEM) micrographs of Ketoconazolee-loaded niosomes.
Determination of Zeta Potential
Zeta potential result of optimized niosomal
suspension was found to be -49.1
High zeta potential prevents the aggregation between the
vesicles hence, enhances physical stability. It has been
investigated that high zeta potential in niosomes increase
inter B layer distance owing to Electrotonic Repulsion.
Figure 11: Zeta potential of Ketoconazole.
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Particle size
Figure 12: FTIR of Particle size of Ketoconazole.
Identification of Ketoconazole
Table 6: Variables and Constrains In Factorial Design.
Independent variables Level
-1 0 1
X1 : Amount of cholesterol (mg) 75 80 160
X2 : Amount of span 60 (mg) 80 100 200
X3 : Amount of drug 10 20 40
Dependent variables
Y1: Entrapment Efficiency
Y2: cumulative Drug Release
Y3: Drug content
Table 7: Experimental design of 32factorial design.
Formulation code X1 X2
F1 -1 -1
F2 -1 0
F3 -1 +1
F4 0 -1
F5 0 0
F6 0 0
F7 0 +1
F8 +1 -1
F9 +1 0
F10 +1 +1
32 factorial design
Table 8: Variables and constrains in 3 – square experimental design.
Independent variables Level
Constrains -1 0 +1
X1 : % of cholesterol 216 322.4 431 In the range
X2 : % of span 60 114 156.4 197 In the range
Dependent variables Constrains
Y1 : Entrapment Efficiency Maximum
Y2 : Drug release Maximum
Y3 : Drug content Minimum
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Optimization Study
A. Table 9. Anova Table of Entrapment efficiency.
Source p-value
Model 0.0017
X1-Cholesterol 0.0229
X2-Span-60 0.0126
X1 X2 0.8483
X1² 0.0117
X2² 0.0004
Figure 13: 3D surface plot for Entrapment efficiency.
Entrapment efficiency % (Y1)=49.54 -1.36X1 – 1.62X2 – 0.094 X1X2 – 2.66 X12 + 6.72 X2
2
B. Table 10. Anova Table For Cumulative Drug Release %.
Source. p-value
Model 0.0122
X1-Cholesterol 0.0014
X2-Span-60 0.1001
X1 X2 0.4756
X1² 0.1121
X2² 0.6602
Figure 14: 3D surface plot for cumulative drug release.
Cumulative drug release %(Y2) = 7.63 – 1.92X1 – 2.3X2 +0.86 X1X2 + 2.95 X1
2 + 0.68 X2
2
C. Table 11. Anova Table For Vesicle Size.
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Source p-value
Model 0.0072
X1-Cholesterol 0.0383
X2-Span-60 0.0008
X1 X2 0.5335
X1² 0.1802
X2² 0.3573
Figure 15: 3D surface plot for Vesicle size.
Mean vesicle size %(Y3) = 5.05 – 22.82 X1 – 66.16X2 +6.24 X1X2 + 19.4 X1
2 + 12.4 X2
2
Table 12: Drug release kinetics.
Formulation Higuchi Korsemeyer peppas Zero order First order Hixson crowel
Ketoconazole r
2 N r
2 r
2 r
2
0.9196 0.3205 0.9531 0.9202 0.9166
Table 13: Stability of the Optimized Ketoconazole loaded niosomes.
Formulation conditions Entrapment efficiency (%)
Ketoconazole
1 mo 2 mo 3 mo
4 °C± 2 °C 49.85 ± 0.7158 48.31±0.7852 48.11± 0.8370
25 °C± 2 °C 47.95± 0.9005 47.20±1.0737 46.32±1.1084
40 °C± 2 °C 45.80± 0.9063 44.33± 0.9525 43.18±0.9756
9. SUMMARY AND CONCLUSION
Niosomes were prepared and evaluated for their abilities
to enhance systemic delivery of Ketoconazole, as
compared to administration of oral tablets. FTIR Studies
revealed that excipients were compatible with the drug.
Pre-formulation study for the drug surfactant
compatibility by FTIR gave conformation about their
purity and showed no interaction between drug and
selected surfactant various formulations were developed
by using non ionic surfactants (cholesterol, span 60)
these are thin film hydration. Developed niosomes were
evaluated for size and shape, surface morphology, drug
entrapment efficiency, in vitro drug release studies,
stability properties. The best entrapment efficiency and
the best in-vitro drug release profile were achieved by
formulations. it was concluded that, Ketoconazole was
successfully encapsulated into niosomes. Several in vitro
and in vivo characterizations of both the drug-loaded
niosomes and films were performed. Ketoconazole was
first entrapped in different niosomal formulations, and
drug-loaded niosomes with small size, low
polydispersity, and high EE were selected for
incorporation into different fast dissolving films, which
were then evaluated for different physical characteristics.
The optimal niosomal film showed sustained release of
the drug compared to the medicated film containing the
free drug. The in vitro release kinetics of drug from the
niosomal suspension and niosomal film followed the
Higuchi diffusion model. Moreover, the in vivo study in
rabbits showed significantly higher rate and extent of
Ketoconazole absorption from sublingual fast dissolving
niosomal film compared to that from oral commercial
tablets. Consequently, the absolute bioavailability of the
drug following sublingual administration was
significantly higher than that after oral tablet
administration. These results indicated that the prepared
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sublingual fast dissolving niosomal film could have
potential as an efficient delivery system to enhance the
bioavailability and prolong the therapeutic effect of
Ketoconazole, thus improving the patient compliance by
eliminating the need for frequent dosing of the drug.
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